Replicating tissue barriers is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Elongated protein‐based micro‐ and nanostructures are of great interest for a wide range of biomedical applications, where they can serve as a backbone for surface functionalization and as vehicles for drug delivery. Current production methods for protein constructs lack precise control of either shape and dimensions or render structures fixed to substrates. This work demonstrates production of recombinant spider silk nanowires suspended in solution, starting with liquid bridge induced assembly (LBIA) on a substrate, followed by release using ultrasonication, and concentration by centrifugation. The significance of this method lies in that it provides i) reproducability (standard deviation of length <13% and of diameter <38%), ii) scalability of fabrication, iii) compatibility with autoclavation with retained shape and function, iv) retention of bioactivity, and v) easy functionalization both pre‐ and post‐formation. This work demonstrates how altering the function and nanotopography of a surface by nanowire coating supports the attachment and growth of human mesenchymal stem cells (hMSCs). Cell compatibility is further studied through integration of nanowires during aggregate formation of hMSCs and the breast cancer cell line MCF7. The herein‐presented industrial‐compatible process enables silk nanowires for use as functionalizing agents in a variety of cell culture applications and medical research.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

We report the fabrication of polymer microstructures with inert surface finish by folding and self-bonding. Layers of off-stoichiometric thiol-ene-epoxy (OSTE+) were fabricated using UV photocuring (1st curing step) of resin, which resulted in a malleable sheet with unreacted groups at its surface. The sheets were structured by either photomasking during the 1st curing step or by cutting postcuring using x urography. The sheets were subsequently folded into a desired shape. Last, the folded structures were thermally cured (2nd curing step), during which surfaces in contact with each other spontaneously bond, the polymer stiffens, the folding stress is released, and reactive surface groups are consumed. We used this unique bend-and-bond technique to demonstrate three sample structures: a multilayer microfluidic chip with crossing channels, a helical spring, and a functional mini windmill. We believe OSTE+ origami can find further applications in the fabrication of biochips and biosensors.

Basement membrane is a thin but dense network of self-assembled extracellular matrix (ECM) protein fibrils that anchors and physically separates epithelial/endothelial cells from the underlying connective tissue. Current replicas of the basement membrane utilize either synthetic or biological polymers but have not yet recapitulated its geometric and functional complexity highly enough to yield representative in vitro co-culture tissue models. In an attempt to model the vessel wall, we seeded endothelial and smooth muscle cells on either side of 470 +/- 110 nm thin, mechanically robust, and nanofibrillar membranes of recombinant spider silk protein. On the apical side, a confluent endothelium formed within 4 days, with the ability to regulate the permeation of representative molecules (3 and 10 kDa dextran and IgG). On the basolateral side, smooth muscle cells produced a thicker ECM with enhanced barrier properties compared to conventional tissue culture inserts. The membranes withstood 520 +/- 80 Pa pressure difference, which is of the same magnitude as capillary blood pressure in vivo. This use of protein nanomembranes with relevant properties for co-culture opens up for developing advanced in vitro tissue models for drug screening and potent substrates in organ-on-a-chip systems.

We show for the first time that 470 nm thick spider silk membranes support co-culturing of endothelial (HDMEC) and smooth muscle cells (SMC). These cell-silk-cell constructs mimic the wall of small blood vessels. The silk membranes are formed through self-assembly at the liquid:air interface of a standing solution of spider silk protein. We show that the silk membranes enable communication between the cells better than commercial tissue culture inserts (TC-inserts).

In this work we present three different ways to characterize the mechanical properties of spider silk nanomembranes. The nanomembranes are formed by self-assembly at the liquid:air interface of a standing solution from which they can be lifted. The mechanical properties are evaluated by (1) manually dropping lead bullets onto the nanomembrane, (2) motorized lowering of a cylindrical indenter to record force-deformation characteristics, and (3) using a standard bulging experiments. Using these methods we show that the nanomembranes are both strong and flexible opening up for applications as pneumatic actuators in MEMS microvalves, or as cell layer actuators in organ-on-a-chip. 

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers.

A method for manufacturing shaped polymers of surface-active macromolecules, in particular silk, is provided. The method is comprising the steps of: • a) depositing an aqueous solution of the surface-active macromolecules on a surface, wherein the aqueous solution of the surface-active macromolecules is deposited in the form of a droplet, and wherein the surface is a hydrophobic micropatterned surface adapted to prevent the aqueous solution from penetrating into the pattern and to receive the droplet of the aqueous solution of the surface-active macromolecules and retain its droplet state; and • b) forming shaped polymers of the surface-active macromolecules on the surface.